Methods of correcting imbalance between bone resorption and bone formation and kits and compositions therefor

ABSTRACT

Compounds, methods, uses, compositions, kits and packages for the treatment of imbalance between bone resorption and bone formation, based on uses of 4-phenyl-2-propionamidotetralin (4-P-PDOT) and analogs, derivatives, prodrugs, precursors thereof, and salts thereof, are described.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation application of U.S. patent application Ser. No.12/526,311, filed on Aug. 7, 2009 now U.S. Pat. No. 8,129,432, which isthe U.S. National Stage Application of PCT application NoPCT/CA2008/000312 filed on Feb. 15, 2008 and published in English underPCT Article 21(2), which itself claims the benefit of U.S. provisionalapplication Ser. No. 60/890,100, filed on Feb. 15, 2007, of U.S.provisional application Ser. No. 60/912,267, filed on Apr. 17, 2007, ofU.S. provisional application Ser. No. 60/915,196 filed on May 1, 2007and of U.S. provisional application Ser. No. 60/938,025 filed on May 15,2007. All documents above are incorporated herein in their entirety byreference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

N/A.

FIELD OF THE INVENTION

The present invention relates to methods of correcting imbalance betweenbone resorption and bone formation, and kits and compositions therefor.

REFERENCE TO SEQUENCE LISTING

Pursuant to 37 C.F.R. 1.821(c), a sequence listing is submitted herewithas an ASCII compliant text file named“765-USPTO-sequence_listing_(—)1403399_ST25” created on Nov. 3, 2011 andhaving a size of ˜2 kilobytes. The content of the aforementioned file ishereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Bones undergo a process of constant remodeling consisting of thebreakdown of old bone and re-building of new bone. This resorption (byosteoclasts) and formation (by osteoblasts) occurs at an approximatelyequal rate thereby maintaining strength of the entire skeleton. Boneremodeling enables the renewal of bone mass and is subjected to theinfluence of a number of hormones and growth factors. It has been shownthat melatonin stimulates bone formation through its action onosteoblasts.

Osteoporosis is defined by the World Health Organization (WHO) in womenas a bone mineral density 2.5 standard deviations below peak bone mass(20-year-old sex-matched healthy person average) as measured by dualenergy X-ray absorptiometry (DXA); the term “established osteoporosis”includes the presence of a fragility fracture.

There are two types of osteoporosis: (1) Primary osteoporosis—bone lossthat occurs as a consequence of the normal aging process and most oftenaffects postmenopausal women and (2) Secondary osteoporosis—bone lossthat occurs as a consequence of other factors such as a chronic medicalcondition, nutritional deficiency, or certain types of medications.

Currently, in the United States, several medications are approved by theU.S. Food and Drug Administration (FDA) for the prevention and treatmentof osteoporosis and are considered as first-line medications. Thesemedications include bisphosphonates, raloxifene, nasal calcitonin andteriparatide.

While treatment modalities are becoming available (such as thebisphosphonates), prevention is still considered the most efficient wayto reduce fracture.

Accordingly, there is a need for novel methods for preventing and/ortreating bone diseases such as osteoporosis.

The present description refers to a number of documents, the content ofwhich is herein incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

The present invention relates to methods of correcting imbalance betweenbone resorption and bone formation, and kits and compositions therefor.

More specifically, in accordance with an aspect of the presentinvention, there is provided a method comprising: (a) identifying asubject suffering from imbalance between bone resorption and boneformation; and (b) administering to said subject a therapeuticallyeffective amount of (i) 4-phenyl-2-propionamidotetralin (4-P-PDOT); (ii)a derivative, analog, conjugate or prodrug of 4-P-PDOT; (iii) apharmaceutical acceptable salt of (i) or (ii); or (iv) any combinationof (i) to (iii), whereby imbalance between bone resorption and boneformation is corrected in said subject.

In an embodiment, the above-mentioned method comprises administering tosaid subject an effective amount of (i) 4-P-PDOT; (ii) a pharmaceuticalacceptable salt of 4-P-PDOT; or (iii) any combination of (i) and (ii).

In an embodiment, the above-mentioned administration is a single bolusadministration. In another embodiment, the above-mentionedadministration is a daily administration.

In an embodiment, the above-mentioned therapeutically effective amountis between about 0.001 and about 500 mg/kg of subject/day.

In an embodiment, the above-mentioned method further comprises theadministration of another agent selected from the group consisting of anMT2 melatonin receptor specific antagonist, a bisphosphonate,raloxifene, nasal calcitonin and teriparatide.

In another aspect, the present invention provides a kit or packagecomprising: (a) at least one compound selected from (i) 4-P-PDOT; (ii) aderivative, analog, conjugate or prodrug of 4-P-PDOT; and (iii) apharmaceutical acceptable salt of (i) or (ii); and (b) instructions toadminister said compound to a subject to correct or prevent bonemineralization defect.

In an embodiment, the above-mentioned kit or package comprises (i)4-P-PDOT; (ii) a pharmaceutical acceptable salt of 4-P-PDOT; or (iii)any combination of (i) and (ii).

In an embodiment, the above-mentioned instructions are instructions toadminister said compound to a subject to correct imbalance between boneresorption and bone formation.

In another embodiment, the above-mentioned kit or package furthercomprises another agent selected from the group consisting of a MT2melatonin receptor specific antagonist, a bisphosphonate, raloxifene,nasal calcitonin and teriparatide.

In another aspect, the present invention provides a compositioncomprising: (a) (i) 4-P-PDOT; (ii) a derivative, analog, conjugate orprodrug of 4-P-PDOT; (iii) a pharmaceutical acceptable salt of (i) or(ii); or (iv) any combination of (i) to (iii); and (b) a therapeuticallyeffective amount of an agent selected from the group consisting of a MT2melatonin receptor specific antagonist, a bisphosphonate, raloxifene,nasal calcitonin and teriparatide.

In another aspect, the present invention provides a composition forcorrecting an imbalance between bone resorption and bone formation in asubject comprising: (a) (i) 4-P-PDOT; (ii) a derivative, analog,conjugate or prodrug of 4-P-PDOT; (iii) a pharmaceutical acceptable saltof (i) or (ii); or (iv) any combination of (i) to (iii); and (b) apharmaceutically acceptable carrier.

In another aspect, the present invention provides a bone targetedcomposition comprises: (a) (i) 4-P-PDOT; (ii) a derivative, analog,conjugate or prodrug of 4-P-PDOT; (iii) a pharmaceutical acceptable saltof (i) or (ii); or (iv) any combination of (i) to (iii); and (b) apharmaceutically acceptable carrier.

In an embodiment, the above-mentioned composition comprises: (a) (i)4-P-PDOT); (ii) a pharmaceutical acceptable salt of 4-P-PDOT; or (iii)any combination of (i) and (ii); and (b) a pharmaceutically acceptablecarrier.

In another aspect, the present invention provides a use of atherapeutically effective amount of (i) 4-P-PDOT; (ii) a derivative,analog, conjugate or prodrug of 4-P-PDOT; (iii) a pharmaceuticalacceptable salt of (i) or (ii); or (iv) any combination of (i) to (iii),in the manufacture of a medicament for the treatment of imbalancebetween bone resorption and bone formation.

In another aspect, the present invention provides a use of atherapeutically effective amount of (i) 4-P-PDOT; (ii) a derivative,analog, conjugate or prodrug of 4-P-PDOT; (iii) a pharmaceuticalacceptable salt of (i) or (ii); or (iv) any combination of (i) to (iii),for the treatment of imbalance between bone resorption and boneformation.

In an embodiment, the above-mentioned use is of (i) 4-P-PDOT; (ii) apharmaceutical acceptable salt of 4-P-PDOT; or (iii) a combination of(i) and (ii).

In an embodiment, the above-mentioned correction of imbalance betweenbone resorption and bone formation comprises at least one of: aninhibition of bone resorption; an inhibition of osteoclastdifferentiation; an increase in bone mineral density (BMD); an increasein bone mineral content (BMC); an increase of density of pure corticalbone; an increase of mean density of total bone; an increase of corticalthickness; an increase of pure cortical area assigned to be cortical; anincrease of tibial diaphyseal total bone areas; an increase ofmineralization apposition rate; an increase of bone formation rate/bonesurface referent; an increase of mineralizing surface for endocorticalor periosteal surface; a decrease of serum alkaline phosphatase; adecrease of intra-cortical regions of hypo-mineralized osteoid; adecrease of osteoid thickness and a decrease of osteoid condensation.

In a further embodiment, the above-mentioned correction of imbalancebetween bone resorption and bone formation comprises an inhibition ofbone resorption. In another embodiment, the above-mentioned correctionof imbalance between bone resorption and bone formation comprises aninhibition of osteoclast differentiation. In another embodiment, theabove-mentioned correction of imbalance between bone resorption and boneformation comprises an increase in bone mineral density (BMD). Inanother embodiment, the above-mentioned correction of imbalance betweenbone resorption and bone formation comprises an increase in bone mineralcontent (BMC).

In an embodiment, the above-mentioned use further comprises the use ofanother agent selected from the group consisting of a MT2 melatoninreceptor specific antagonist, a bisphosphonate, raloxifene, nasalcalcitonin and teriparatide.

In another aspect, the present invention provides a method comprising:(a) identifying a subject suffering from imbalance between boneresorption and bone formation; and (b) administering a therapeuticallyeffective amount of at least one MT2 melatonin receptor specificantagonist to the subject, whereby imbalance between bone resorption andbone formation is corrected in the subject.

In another aspect, the present invention provides a use of atherapeutically effective amount of at least one MT2 melatonin receptorspecific antagonist in the manufacture of a medicament for the treatmentof imbalance between bone resorption and bone formation.

In another aspect, the present invention provides a use of atherapeutically effective amount of at least one MT2 melatonin receptorspecific antagonist in the treatment of imbalance between boneresorption and bone formation.

The present invention further provides a composition for the treatmentof imbalance between bone resorption and bone formation, saidcomposition comprising at least one MT2 melatonin receptor specificantagonist and a pharmaceutically acceptable carrier.

The present invention further provides a kit or package comprising atleast one MT2 melatonin receptor specific antagonist and instructionsfor the treatment of imbalance between bone resorption and boneformation.

In an embodiment, the above-mentioned subject suffers from osteoporosis.In another embodiment, the above-mentioned subject suffers from Pagetdisease. In another embodiment, the above-mentioned subject suffers fromosteolytic bone cancer. In another embodiment, the above-mentionedsubject suffers from arthritis characterized by the presence of aninflammatory cytokine that induces osteoclasts.

In an embodiment, the above-mentioned subject is a mammal. In a furtherembodiment, the above-mentioned subject is a human.

As used herein the term “subject” is meant to refer to any mammalincluding human, mice, rat, dog, cat, pig, monkey, horse, etc. In aparticular embodiment, it refers to a human.

The articles “a,” “an” and “the” are used herein to refer to one or tomore than one (i.e., to at least one) of the grammatical object of thearticle.

The term “including” and “comprising” are used herein to mean, and areused interchangeably with, the phrases “including but not limited to”and “comprising but not limited to”.

The terms “such as” are used herein to mean, and are usedinterchangeably with, the phrase “such as but not limited to”.

By “agonist” it is meant that the ligand stimulates a ligand-dependentreceptor-characteristic activity above any baseline levels present inthe absence of ligand. By “antagonist” it is meant that the ligand bindsto the receptor and functions as a competitive or non-competitiveinhibitor of receptor-characteristic agonist activity. By “inverseagonist” or “reverse agonist” it is meant that the ligand will bind tothe receptor in question and cause the suppression of receptor activitylower than the amount of activity seen in the absence of receptorligand. As used herein the terms “MT2 melatonin receptor specificantagonist” are meant to refer to an antagonist that binds specificallyto the MT2 receptor as opposed to other antagonists which bind to othermelatonin receptors such as luzindole.

As used herein the terms “osteoclast precursor” are meant to refer to acell that is able to mature into an osteoclast. Without being solimited, such cell includes RAW264.7, spleen cells, haematopoietic cellsable to mature into osteoclasts and CD14⁺ monocytes.

There are a number of agents known to provoke the differentiation of anosteoclast precursor into an osteoclast. Without being so limited, theyinclude Receptor Activator for Nuclear Factor Kappa B Ligand (RANKL),macrophage-colony stimulating factor (M-CSF), inflammatory cytokinessuch as tumor necrosis factor-alpha (TNF-α) and various interleukinsable to stimulate osteoclast activity.

There are a number of resorbable bone analogs available commerciallysuch as but not limited to BioCoat™. Other resorbable bone analogsinclude dentine fragment and hydroxyapatite.

There are a number of known animal models for imbalance between boneresorption and bone formation including C57Bl/6j mice, which is known toexhibit a very low bone mineral density (BMD), and osteoporosis animalmodels.

There are a number of known osteoporosis animal models includingovariectomized mice and rats.

Included within the scope of the subject invention are derivatives,analogs, conjugates, or prodrugs of 4-phenyl-2-propionamidotetralin(4-P-PDOT), and salts thereof, which have the ability, as describedherein, to treat an imbalance between bone resorption and bone formationin a subject. The salt(s) mentioned herein include pharmaceuticallyacceptable salt(s). Various analogs, derivatives, conjugates andprodrugs of 4-P-PDOT are known and include, for example,8-methoxy-2-propionamido-tetralin; 2-chloroacetamido-tetralin;8-methoxy-2-n-butyramido-tetralin;8-methoxy-2-cyclopropanecarbonylamido-tetralin;8-methoxy-2-chloroacetamido-tetralin; 4-phenyl-2-acetamido-tetralin(4-P-ADOT); 4-benzyl-2-acetamidotetralin;4-phenyl-2-chloroacetamido-tetralin (4-P-CADOT); and4-benzyl-2-propionamido-tetralin, described in U.S. Pat. No. 5,071,875.In an embodiment, the above-mentioned derivative is 4-P-ADOT or4-P-CADOT.

Kits

The present invention also relates to a kit or package fortreating/preventing imbalance between bone resorption and bone formation(e.g. for inhibiting bone resorption or osteoclastmaturation/differentiation) comprising at least one compound selectedfrom (i) 4-phenyl-2-propionamidotetralin (4-P-PDOT); (ii) a derivative,analog, conjugate or prodrug of 4-P-PDOT; and (iii) a pharmaceuticalacceptable salt of (i) or (ii); and instructions to administer saidcompound to a subject to inhibit inhibiting bone resorption orosteoclast maturation/differentiation. Such kits may also comprise acomposition (e.g. a pharmaceutical composition) comprising at least oneof the above-mentioned compounds and a pharmaceutically acceptablecarrier. Such kits may further comprise at least one other active agentable to inhibit bone resorption or osteoclastmaturation/differentiation. When the kit is used to inhibit boneresorption or osteoclast maturation/differentiation in a subject havingosteoporosis, the kit may also further comprise at least one otheractive agent capable of preventing or correcting any other detrimentalsymptoms of osteoporosis. Such agents include without being so limitedbisphosphonates, raloxifene, nasal calcitonin and teriparatide. Inaddition, a compartmentalized kit in accordance with the presentinvention includes any kit in which reagents are contained in separatecontainers. Such containers include small glass containers, plasticcontainers or strips of plastic or paper. Such containers allow theefficient transfer of reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another.

As used herein the term “imbalance between bone resorption and boneformation” is meant to refer to an increase in the rate of osteoclastmaturation or differentiation, generating more mature osteoclasts aswell as an increase of resorption activity by mature osteoclasts.

As used herein the term “correct” when used in the context of correctionof an imbalance between bone resorption and bone formation is meantherein to refer to any partial or complete improvement of imbalancebetween bone resorption and bone formation. Such correction maycorrespond to, without being so limited, an inhibition of boneresorption, an inhibition of osteoclast differentiation, an increase inbone mineral density (BMD) and bone mineral content (BMC); an increaseof density of pure cortical bone, an increase of mean density of totalbone, an increase of cortical thickness, an increase of pure corticalarea assigned to be cortical, an increase of tibial diaphyseal totalbone areas, an increase of mineralization apposition rate, an increaseof bone formation rate/bone surface referent, an increase ofmineralizing surface for endocortical or periosteal surface; a decreaseof serum alkaline phosphatase, a decrease of intra-cortical regions ofhypo-mineralized osteoid, a decrease of osteoid thickness and a decreaseof osteoid condensation.

Route of Administration

Pharmaceutical compositions of the present invention can be administeredby routes such as orally, nasally, intravenously, intramuscularly,subcutaneously, sublingually, intrathecally, or intradermally. The routeof administration can depend on a variety of factors, such as theenvironment and therapeutic goals.

By way of example, pharmaceutical composition of the invention can be inthe form of a liquid, solution, suspension, pill, capsule, tablet,gelcap, powder, gel, ointment, cream, nebulae, mist, atomized vapor,aerosol, or phytosome. For oral administration, tablets or capsules canbe prepared by conventional means with pharmaceutically acceptableexcipients such as binding agents, fillers, lubricants, disintegrants,or wetting agents. The tablets can be coated by methods known in theart. Liquid preparations for oral administration can take the form of,for example, solutions, syrups, or suspension, or they can be presentedas a dry product for constitution with saline or other suitable liquidvehicle before use. Dietary supplements of the invention also cancontain pharmaceutically acceptable additives such as suspending agents,emulsifying agents, non-aqueous vehicles, preservatives, buffer salts,flavoring, coloring, and sweetening agents as appropriate. Preparationsfor oral administration also can be suitably formulated to givecontrolled release of the active ingredients.

Dosage

Any amount of a pharmaceutical composition can be administered to asubject. The dosages will depend on many factors including the mode ofadministration and the age of the subject. In younger people there isextensive bone-turnover due to growing bone. Typically, the amount of acompound or agent of the present invention (e.g., 4-P-PDOT, aderivative, analog, conjugate or prodrug of 4-P-PDOT; a pharmaceuticalacceptable salt thereof) contained within a single dose will be anamount that effectively prevent, delay or correct bone resorption in asubject in need thereof without inducing significant toxicity. As usedherein the term “therapeutically effective amount” is meant to refer toan amount effective to achieve the desired therapeutic effect. Atherapeutically effective amount is also one in which any adverse sideeffects of the compound are outweighed by the therapeutically beneficialeffects. Typically, a compound or agent of the present invention (e.g.,4-P-PDOT, a derivative, analog, conjugate or prodrug of 4-P-PDOT; or apharmaceutical acceptable salt thereof) can be administered to subjectsin doses ranging from 0.001 to 500 mg/kg/day and, in a more specificembodiment, 1 mg to 5 mg/kg/day. The allometric scaling method ofMahmood et al. (J. Clin. Pharmacol. 2003, 43(7): 692-7) can be used toextrapolate the dose from mice to human. Pharmaceutically acceptablepreparations and salts of the small molecules of the present inventionare within the scope of the present invention and are well known in theart (Remington's Pharmaceutical Science, 16th Ed., Mack Ed.). The dosagewill be adapted by the clinician in accordance with conventional factorssuch as the extent of the disease and different parameters from thepatient.

The therapeutically effective amount of a compound or agent of thepresent invention (e.g., 4-P-PDOT, a derivative, analog, conjugate orprodrug of 4-P-PDOT; or a pharmaceutical acceptable salt thereof) mayalso be measured directly. The effective amount may be given daily orweekly or fractions thereof. Typically, a pharmaceutical composition ofthe invention can be administered in an amount from about 0.001 mg up toabout 500 mg per kg of body weight per day (e.g., 1 mg, 2 mg, 3 mg, 4mg, 5 mg, 10 mg, 50 mg, 100 mg, or 250 mg). Dosages may be provided ineither a single or multiple dosage regimen(s). For example, in someembodiments the effective amount is a dose that ranges from about 1 mgto about 25 grams of the agent per day, about 50 mg to about 10 grams ofthe agent per day, from about 100 mg to about 5 grams of the agent perday, about 1 gram of the agent per day, about 1 mg to about 25 grams ofthe agent per week, about 50 mg to about 10 grams of the agent per week,about 100 mg to about 5 grams of the agent every other day, and about 1gram of the agent once a week.

These are simply guidelines since the actual dose must be carefullyselected and titrated by the attending physician based upon clinicalfactors unique to each patient. The optimal daily dose will bedetermined by methods known in the art and will be influenced by factorssuch as the age of the patient as indicated above and other clinicallyrelevant factors. In addition, patients may be taking medications forother diseases or conditions. The other medications may be continuedduring the time that the agent is given to the patient, but it isparticularly advisable in such cases to begin with low doses todetermine if adverse side effects are experienced.

Carriers/Vehicles

The compound or agent of the present invention (e.g., 4-P-PDOT, aderivative, analog, conjugate or prodrug of 4-P-PDOT; or apharmaceutical acceptable salt thereof) may be incorporated into dosageforms in conjunction with any of the vehicles which are commonlyemployed in pharmaceutical preparations, e.g., talc, gum arabic,lactose, starch, magnesium searate, cocoa butter, aqueous or non-aqueoussolvents, oils, paraffin derivatives or glycols. Emulsions such as thosedescribed in U.S. Pat. No. 5,434,183 may also be used in which vegetableoil (e.g., soybean oil or safflower oil), emulsifying agent (e.g., eggyolk phospholipid) and water are combined with glycerol. Furthernon-limiting pharmaceutically suitable materials that may beincorporated in pharmaceutical preparations of the present inventioninclude absorption enhancers, pH regulators and buffers, osmolarityadjusters, preservatives, stabilizers, antioxidants, surfactants,thickeners, emollient, dispersing agents, flavoring agents, coloringagents and wetting agents. Methods for preparing appropriateformulations are well known in the art (see e.g., Remington'sPharmaceutical Sciences, 16th Ed., 1980, A. Oslo Ed., Easton, Pa.).

In cases where parenteral administration is elected as the route ofadministration, preparations containing the agent may be provided topatients in combination with pharmaceutically acceptable sterile aqueousor non-aqueous solvents, suspensions or emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oil, fish oil, and injectable organic esters. Aqueous carriersinclude water, water-alcohol solutions, emulsions or suspensions,including saline and buffered medical parenteral vehicles includingsodium chloride solution, Ringer's dextrose solution, dextrose plussodium chloride solution, Ringer's solution containing lactose, or fixedoils. Intravenous vehicles may include fluid and nutrient replenishers,electrolyte replenishers, such as those based upon Ringer's dextrose,and the like.

In yet another embodiment, the pharmaceutical compositions of thepresent invention can be delivered in a controlled release system. Inembodiments, polymeric materials including polylactic acid,polyorthoesters, cross-linked amphipathic block copolymers andhydrogels, polyhydroxy butyric acid and polydihydropyrans can be used(see also Smolen and Ball, Controlled Drug Bioavailability, Drug productdesign and performance, 1984, John Wiley & Sons; Ranade and Hollinger,Drug Delivery Systems, pharmacology and toxicology series, 2003, 2^(nd)edition, CRRC Press), in another embodiment, a pump may be used (Saudeket al., 1989, N. Engl. J. Med. 321: 574). Compounds of the presentinvention may also be delivered by the use of targeting molecules and/ormoiety (e.g., a monoclonal antibody, a peptide) as individual carriersto which the agent is coupled/associated. In an embodiment, theabove-mentioned targeting molecule/moiety increases and/or facilitatesthe delivery of the agent to the bone (i.e. bone-targetingmolecule/moiety). The present invention also encompasses compoundsmodified to increase their solubility and/or their circulatory time,such as their pegylation.

As used herein, “subject” or “subject in need thereof” refers to animalssuch as humans in which prevention, delay or treatment of a boneresorption defect is desirable. In particular embodiments, subjectshaving diseases or conditions such as osteoporosis, Paget disease andosteolytic bone cancer would benefit from the compounds, compositions,methods and uses of the present invention.

Other objects, advantages and features of the present invention willbecome more apparent upon reading of the following non-restrictivedescription of specific embodiments thereof, given by way of exampleonly with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

In the appended drawings:

FIG. 1 presents RT-PCR results showing that only Mel1b(MT2) is expressedin RAW264.7 cells. Brain tissue served as positive controls (+control);

FIGS. 2A-2F present differentiated RAW264.7 cells positively stained forMT2 receptor. Panel A: Negative control without primary antibody. PanelC: Cells stained with MT2 antibody illustrating the membranelocalization of the protein. Panels B and D: DAPI nuclear stain of bothnegative control and MT2 positive cells. Panels E and F: Negativecontrol (incubation in the presence of a peptide recognized by theantibody and competing with the MT2receptor) and DAPI nuclear stain ofthis negative control, respectively;

FIGS. 3A-B present the number of TRAP-positive RAW264.7 cells. The cellswere cultured with RANKL for 2.5 (panel A) or 6 (panel B) days with(white bars) and without (black bars) 10⁻⁶M of 4-P-PDOT. Raw counts(upper panel) and % of control cells (lower panel) are indicated. Theseresults represent the average of 3 independent experiments;

FIGS. 4A-4C present bone resorption results. Panel A shows the number ofresorption pits, panel B shows the resorbed area (μm²*1000) and panel Cshows the total resorbed area (number of pits X resorbed area(μm²*1000)). RAW264.7 cells were cultured with RANKL for 10 days without(black bars), and with 10⁻⁶M of 4-P-PDOT for 4 (white bars) or 10(hatched bars) days. Results of eight microscopic fields counted induplicate are illustrated. Results summarize 2 independent experiments;

FIGS. 5A-5B present the effect of melatonin on osteoclastsdifferentiation. RAW264.7 cells were cultured with RANKL for 2.5 (panelA) or 6 (panel B) days without (black bars), with 10⁻⁹M of melatonin(white bars) and with melatonin +10⁻⁶M 4-P-PDOT (grey bars). Raw counts(upper panel) and % of control cells (lower panel) are indicated. Theseresults represent the average of 4 independent experiments;

FIG. 6 compares the differentiation of RAW264.7 cells cultured withRANKL for 6 days with 10⁻⁹M of melatonin, 10⁻⁹M melatonin +10⁻⁶M4-P-PDOT, or 10⁻⁶M 4-P-PDOT in terms of percentage of TRAP-positivecells over control (cells treated with RANKL only). The upper panelpresents microscopic fields showing cells after each treatment. Theseresults represent the average of 3 to 6 independent experiments;

FIGS. 7A-7C present the effect of melatonin on osteoclasts function.RAW264.7 cells were cultured with RANKL for 10 days without (blackbars), with 10⁻⁹M of melatonin (white bars), or with 10⁻⁹M of melatonin+10⁻⁶M of 4-P-PDOT (grey bars) for 4 or 10 days. Number of resorptionpits (panel A), resorbed area (μm²*1000) (panel B) and total resorbedarea (number of pits X resorbed area (μm²*1000)) (panel C) of eightmicroscopic fields counted in duplicate are illustrated. These resultssummarize 2 independent experiments;

FIG. 8 compares the differentiation of RAW264.7 cells cultured withRANKL for 10 days with 10⁻⁹M of melatonin, 10⁻⁹M melatonin +10⁻⁶M4-P-PDOT, or 10⁻⁶M 4-P-PDOT in terms of percentage of number ofresorption pits (middle panel) or total resorbed area (lower panel) overcontrol (cells treated with RANKL only). The upper panel presentsmicroscopic fields showing cells after each treatment. These resultssummarize 2 independent experiments;

FIGS. 9A-9C show the effect of increasing concentrations of melatonin(panel A), 4-P-PDOT (panel B) or both (panel C) on adenylyl cyclaseactivity of forskolin-stimulated RAW 264.7 cells;

FIGS. 10A-10C show the normalized cAMP values of forskolin-stimulatedRAW 264.7 cells resulting from increasing concentrations of melatonin(upper panel, A), 4-P-PDOT (middle panel, B) or both (lower panel, C);

FIGS. 11A-11F show a comparison of cell apoptosis in differentiated RAW264.7. Negative control without primary antibody (panel A).DNase-treated cells as positive controls for apoptosis (panel B). Cellstreated with RANKL only (panel C), 4-P-PDOT 10⁻⁶M (panel D), Melatonin10⁻⁹M (panel E) and Melatonin 10⁻⁹M+4-P-PDOT 10⁻⁶M (panel F);

FIG. 12 presents tritiated-thymidine incorporation in RAW264.7 cellstreated with RANKL only, 10⁻⁷M (+mel-7) or 10⁻⁹M (+mel-9) melatonin,10⁻⁶M luzindole (Luz-6), 10⁻⁶M 4-P-PDOT (PP-6), 10⁻⁹M melatonin +10⁻⁸Mluzindole (+mel-9+Luz-8) or 10⁻⁹M melatonin +10⁻⁶M 4-P-PDOT(+mel-9+PP-6);

FIG. 13 presents the frequency of the number of RAW264.7 nucleisubjected to RANKL only, 10⁻⁹M melatonin (mel-9), 10⁻⁶M 4-P-PDOT (PP-6),or 10⁻⁹M melatonin +10⁻⁶M 4-P-PDOT (+mel-9+PP-6);

FIG. 14 presents RANK cDNA expression on RAW264.7 cells cultured withRANKL for 10 days with 10⁻⁹M of melatonin, 10⁻⁹M melatonin +10⁻⁶M4-P-PDOT, or 10⁻⁶M 4-P-PDOT in terms of percentage of expression(normalized over β-actin expression) over control (lower panel). Theupper panel presents RANK cDNA expression (following RT-PCR) on anagarose gel in cells following the different treatments. These resultssummarize 3 independent experiments;

FIG. 15 presents the weight gain variations from day 0 to day 35 incontrol and treated mice;

FIGS. 16A-16B present bone mineral content (BMC), bone area, and bonemineral density (BMD) variations from day 0 to day 35 in whole body ofcontrol and treated mice;

FIGS. 17A-17B present BMC, bone area, and BMD variations from day 0 today 35 in spine of control and treated mice;

FIGS. 18A-18C present BMC, bone area, and BMD variations from day 0 today 35 in left and right femurs of control and treated mice;

FIGS. 19A-19B present TRAP staining on right femur section of maleC57Bl6/j mice. Panel A represents a right femur section of a controlmouse (untreated). Panel B represents a right femur section of a mousetreated with 4-P-PDOT at 10 mg/kg of body weight. There is a significantreduction in the number of osteoclasts in mice treated with 4-P-PDOT.Magnification 20×;

FIGS. 20A-20F present Goldner staining of right femur sections of maleC57Bl/6j mice. Upper panels (A, B and C) represent histological sectionsof untreated mice (n=3, controls), while lower panels (D, E and F)represent histological sections of mice treated with 4-P-PDOT (n=3).Trabecular bone is increased in animal treated with 4-P-PDOT (10 mg/kgof body weight IP injection three times per week); and

FIG. 21 shows the effect of 4-P-PDOT on the number of total osteoclastsvs. active osteoclasts. Total osteoclast numbers were determined onGoldner stained sections while active osteoclasts were calculated basedon the number of TRAP⁺ cells.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention is illustrated in further detail by the followingnon-limiting examples.

Cellular model RAW264.7 (mouse macrophage cell line, ATCC # TIB-71).These cells differentiate in osteoclast-like cells upon addition ofreceptor activator of nuclear factor-kappaB ligand (RANKL). Celldifferentiation usually occurs within two days. Cells were studied at2.5 (early stage), 6 and 10 days (late stage) following RANKL addition.

Experimental treatments: In some experiments, 4-P-PDOT was added to thecells at the same time as RANKL at a concentration of 10⁻⁶M. Cellscultured with RANKL only served as controls.

EXAMPLE 1 Gene and Protein Expression of Melatonin Receptors on RAW264.7Cells

In order to determine the presence of melatonin receptors on RAW 264.7cells, gene and protein expression was examined. RAW264.7 cells werecultured with or without RANKL for a period of 6 days. RNA was extractedusing Trizol™ reagent according to the manufacturer's instructions. 2 μgof total RNA were reverse transcribed using Thermoscript™ RT-PCR system(InVitrogen). PCR reactions for melatonin receptors 1a (MT1) and 1b(MT2) were set up using intron spanning primers. MT2 Forward:5′-GCAGGTAATTTGTTTGTGGT-3′ (SEQ ID NO: 1); MT2 Reverse:5′-AGATGCGTGGATCATACTCT-3′ (SEQ ID NO: 2); MT1 Forward:5′-TGTACCGCAACAAGAAGCTCAGGA-3′ (SEQ ID NO: 3); MT1 Reverse:5′-TGGCGATGAGTGTCAGCATCCATA-3′ (SEQ ID NO: 4). RAW264.7 samples werecompared to a brain tissue sample that was positive for both receptors.The results are presented in FIG. 1. MT2 was thus detected while MT1 wasnot.

MT2 receptor localization was then investigated by immunofluorescence.RAW 264.7 cells were grown for three days with or without RANKL inLabTek™ chamber. Cells were fixed in 3.7% of paraformaldehyde andpermeabilized with 0.1% Triton™-X-100. Cells were then incubated in PBSadded with 1% bovine albumin (PBSA) for 30 minutes before a two-hourincubation with 1:25 dilution of anti-melatonin 1b in PBSA at 37° C.Negative controls were incubated with PBSA alone. After four rinses inPBS, cells were incubated one hour with 1:500 dilution of donkeyanti-goat conjugated to Alexa™ 594 fluorochrome at 37° C. Cells weremounted and images visualized with 40× objective using a microscopeequipped with fluorescence. Exposure times for each color were the samefor all conditions. The results are presented in FIG. 2.

EXAMPLE 2 Effect of 4-P-PDOT on Osteoclast Differentiation as Measuredby Tartrate Resistant Acid Phosphatase (TRAP) Activity

In order to assess osteoclastogenesis, RAW264.7 cells were cultured withRANKL for 2.5 or 6 days with and without 10⁻⁶M of 4-P-PDOT. RAW264.7cells were fixed with paraformaldehyde and stained for TRAP activity(marker of active osteoclasts) at 2.5 and 6 days of differentiation.Positive-stained cells with more than 3 nuclei were counted in eightdifferent microscopic fields done in quadruplicate. Results arepresented in FIGS. 3A and B for the 2.5 or 6 days experiments,respectively. The data show that addition of 4-P-PDOT during RAW264.7cell differentiation reduces the number of TRAP+ cells.

EXAMPLE 3 Osteoclast Function: Bone Resorption Assay

Osteoclast activity was determined using a resorbable bone analog(BioCoat™ Osteologic™ discs, BD Biosciences). RAW 264.7 cells wereplated on 16-well plates coated with this analog. 24 hours post-plating,cells were treated with RANKL alone or with RANKL+10⁻⁶M of 4-P-PDOT fora period of 10 days. In another set of experiments, 10⁻⁶M of 4-P-PDOTwas added after 6 days of culture with RANKL and cells were cultured foran additional 4 days in order to examine the effects of 4-P-PDOT on moremature cells. At the end of the culture period, cells were detached withbleach and bone analog stained with 5% silver nitrate according tomanufacturer's instructions to reveal the presence of resorbed bone area(resorption pits). The number of pits and the area of resorbed bone werequantified in 8 microscopic fields in duplicate using Bioquant™software. Results are presented in FIG. 4. Additional results arepresented in FIG. 8.

EXAMPLE 4 Comparison of Effect on Osteoclasts Differentiation andFunction in Melatonin and 4-P-PDOT

The effect of melatonin on osteoclastogenesis and osteoclast functionwas assessed with the same methods as those described in Examples 2 and3 above except that cells were grown in the presence of 10⁻⁹M ofmelatonin in addition to RANKL. In some experiments, melatonin was addedalong with 10⁻⁶M of 4-P-PDOT. Cells treated with RANKL alone served ascontrols. Addition of melatonin did not have a significant effect onosteoclastogenesis (FIGS. 5A and 5B and FIG. 6) but significantlyreduced bone resorption (FIG. 7).

EXAMPLE 5 Comparison of cAMP Production Induced by Melatonin and4-P-PDOT

RAW264.7 cells were grown in culture medium without RANKL untilconfluence (7 days). Cells were then incubated with 10⁻⁴M of forskolinin presence of various concentrations of melatonin (FIG. 9, panel A),4-P-PDOT (FIG. 9, panel B) or both (FIG. 9, panel C). Concentrationsranged from 10⁻¹¹ to 10⁻⁵M for melatonin and from 10⁻¹° to 10⁻⁴M for4-P-PDOT. In the double treatment group, cells were treated withincreasing concentrations of melatonin (10⁻¹¹ to 10⁻⁵M) to which 10⁻⁶Mof 4-P-PDOT was added. After 30 minutes incubation at 37° C., cells werelysed in TRIS-EDTA buffer supplemented with protease andphosphodiesterase inhibitors at 4° C. The cAMP content was determined induplicate in 200 μL aliquot of the supernatant using an enzymeimmunoassay kit.

FIG. 10 presents normalized cAMP activity values in RAW 264.7 cellstreated with melatonin, 4-P-PDOT or melatonin +4-P-PDOT wherein in eachof panels A, B and C, dose 1 corresponds to the cAMP value for forskolinalone; dose 2 corresponds to 10⁻¹¹ of melatonin, 10⁻¹⁰4-P-PDOT, or acombination of 10⁻¹¹ of melatonin and 10⁻⁶4-P-PDOT, respectively; dose 3corresponds to 10⁻⁹ of melatonin, 10⁻⁸4-P-PDOT, or a combination of 10⁻⁹of melatonin and 10⁻⁶4-P-PDOT, respectively; dose 4 corresponds to 10⁻⁷of melatonin, 10⁻⁶4-P-PDOT, or a combination of 10⁻⁷ of melatonin and10⁻⁶4-P-PDOT, respectively; and dose 5 corresponds to 10⁻⁵ of melatonin,10⁻⁴ 4-P-PDOT, or a combination of 10⁻⁵ of melatonin and 10⁻⁶4-P-PDOT,respectively. cAMP values were normalized against total proteinconcentration which was determined using the Bradford protein assay.

As may be seen from FIGS. 9 and 10, measurement of cAMP productionshowed that these cells respond differentially to melatonin and4-P-PDOT. In the presence of increasing doses of 4-P-PDOT, cAMPproduction increases while this production is inhibited with melatoninincreasing doses.

RAW 264.7 cells then were grown for six days in LabTek™ chamber withRANKL alone or RANKL with melatonin (10⁻⁹M), melatonin (10⁻⁹M) and4-P-PDOT (10⁻⁶M) or 4-P-PDOT (10⁻⁶M) alone. Cells were fixed in 3.7% ofparaformaldehyde and permeabilized with 0.1% Triton™-X-100. Cells werethen incubated in PBS supplemented with 1% bovine albumin (PBSA) for 30minutes before a 30 minute-incubation with 1:40 anti-phalloidin dilutedin PBSA at 37° C. After four rinses in PBS, cells were mounted with amedium containing DAPI and images visualized with 63× objective using amicroscope equipped with fluorescence. Images were taken from 10independent microscopic fields and nuclei counted in multinucleatedcells (more than two nuclei). FIG. 13 presents the frequency of numberof nucleus as % of total multinucleated.

EXAMPLE 6 Comparison of Apoptosis in Treated and Untreated Cells

RAW264.7 cells were grown in culture medium with RANKL for 6 days withor without 10⁻⁶M of 4-P-PDOT. Cells were fixed in 3.7% ofparaformaldehyde and permeabilized with 0.2% Triton™-X-100. A subset ofcells were treated with DNase for 10 minutes at room temperature andserved as positive controls. Cell apoptosis was determined using theDeadEnd™ fluorometric TUNEL system. This method measures fragmented DNAthrough the incorporation of fluorescent-labeled d-UTP. Permeabilizedcells were incubated in equilibration buffer with a nucleotide mix andthe enzyme that catalyzes the reaction for 1 hour at 37° C. Negativecontrols were incubated without the enzyme. After several rinses in SSCand PBS, cells were mounted and visualized by fluorescence microscopy.Results are presented in FIG. 11.

EXAMPLE 7 Comparison of Cell Proliferation in Treated and UntreatedCells

After 24 hour attachment, RAW264.7 cells were serum-starved overnightand then grown for 2.5 days in culture medium containing RANKL andmelatonin (10⁻⁷ or 10⁻⁶M), or melatonin (10⁻⁶M) and a melatonin receptornon-specific antagonist, luzindole (10⁻⁸M), or melatonin (10⁻⁶M) and4-P-PDOT (10⁻⁶M), or luzindole (10⁻⁶M), or 4-P-PDOT (10⁻⁶M) alone.Thymidine incorporation was assayed by the addition of 0.02 μCi/μL oftritiated thymidine to the medium 8 hours before cell harvest. Cellswere then washed twice with ice-cold PBS and three times with cold 5%trichloroacetic acid. Cells were lysed in a mixture of 0.5N NaOH and0.5% SDS and radioactivity of the lysates determined using abeta-counter. Results are presented in FIG. 12.

FIGS. 11 and 12 show that effect of 4-P-PDOT on osteoclast function/boneresorption are not explained by increased apoptosis or by reduced cellproliferation, since these parameters were comparable between4-P-PDOT-treated vs. untreated cells.

EXAMPLE 8 Expression of Osteoclasts Cells Markers

The effect of melatonin and of 4-P-PDOT on the expression of variousgenes involved in osteoclast differentiation and activity was evaluatedby RT-PCR after 6 days of treatment. FIG. 14 shows that melatonin and4-P-PDOT induce a significant decrease of RANK's expression on RAW264.7cell-derived osteoclasts.

EXAMPLE 9 Effect of 4-P-PDOT on Animal Model for Imbalance Between BoneResorption and Bone Formation

3 week-old male C57Bl/6j mice, which are known to exhibit a very lowbone mineral density (BMD) because of a natural mutation limiting theproduction of melatonin, were injected intraperitoneally with 10 mg/kgof 4-P-PDOT three times a week for 1 month. In parallel, control animalsreceived injections of a solution made of water and ethanol (20%).

During this period, animals were subjected to weekly bone densitometricmeasurements (BMD and bone mineral content (BMC)) with a PixiMus™ IIbone densitometer, and blood and urine collection. Scan of individualspine, long bones (femurs), were performed. Scans of tibiae, radius andmandible were also performed.

To monitor bone formation rate, 25 mg/kg of tetracycline hydrochloridewas injected on days 10 and 20 following the beginning of theinjections. Bone histomorphometry for bone formation and resorptionstatic and dynamic parameters were also measured. The biochemicalmarkers used are alkaline phosphatase for bone formation, and urinarydeoxypyridinoline for bone resorption.

The first 35 days of the experiments revealed an increase in weightgain, bone mineral accretion at the spine and whole body level intreated mice compared to vehicle-injected animals (see FIGS. 16-18).

The analysis is continued over 12 months. After 6 or 12 months, mice aresacrificed and individual bones and spine scanned by MicroCT™ (SkyScanCT analyzer) and subsequently fixed and embedded in methylmetacrylateresin to perform histomorphometry analyzes (which include, surface andvolume of bone cells: ostoids, osteoblasts, osteocytes, osteoclasts,thickness of cortex and trabeculaes).

The effect of 4-P-PDOT on the formation of bones has also been testedusing 4-week old male C57BL/6 mice following weaning. Mice were injectedintraperitoneally with 10 mg/kg of 4-P-PDOT three times a week for 4weeks (28 days). After this period, femur sections were stained toquantify tartrate-resistant acidic phosphatase- (TRAP-) positiveosteoclasts (i.e. “active” osteoclasts). FIG. 19 shows that mice treatedwith 4-P-PDOT have a significant reduction in the number of activeosteoclasts as compared to control mice. A Goldner staining was alsoperformed on the femur sections in order to quantify the total number ofosteoclasts. As shown in FIG. 20, treatment with 4-P-PDOT also resultsin an increased in the number of total osteoclasts and in trabecularbone. Also, the results presented in FIG. 21 clearly indicate that micetreated with 4-P-PDOT show a significant reduction (about 3-fold) in therelative number of active osteoclasts in vivo (although the relativenumber of total osteoclasts is increased) as compared to vehicle-treatedmice.

Although the present invention has been described hereinabove by way ofspecific embodiments thereof, it can be modified, without departing fromthe spirit and nature of the subject invention as defined in theappended claims.

1. A composition comprising (a) (i) 4-P-PDOT; (ii) a conjugate of4-P-PDOT; (iii) a pharmaceutical acceptable salt of (i) or (ii); or (iv)any combination of (i) to (iii); and (b) a therapeutically effectiveamount of an agent that is a bisphosphonate, raloxifene, nasalcalcitonin or teriparatide.
 2. The composition of claim 1, comprising(a) (i) 4-P-PDOT; (ii) a pharmaceutical acceptable salt of 4-P-PDOT; or(iii) any combination of (i) and (ii) and (b) a therapeuticallyeffective amount of an agent that is a bisphosphonate, raloxifene, nasalcalcitonin or teriparatide.
 3. A kit comprising (a) at least onecompound selected from (i) 4-P-PDOT; (ii) conjugate of 4-P-PDOT; and(iii) a pharmaceutical acceptable salt of (i) or (ii); (b) another agentthat is bisphosphonate, raloxifene, nasal calcitonin or teriparatide;and (c) instructions to administer said compound to a subject to treat abone mineralization defect.
 4. The kit of claim 3, wherein the kitcomprises (i) 4-P-PDOT; (ii) a pharmaceutical acceptable salt of4-P-PDOT; or (iii) any combination of (i) and (ii).
 5. The kit of claim4, wherein said instructions are instructions to administer saidcompound to a subject to improve an imbalance between bone resorptionand bone formation.